The use of antibodies to interfere with the normal function of gonadrotrophin releasing hormone (GnRH, also known as LHRH or lutenizing hormone releasing hormone) in mammals in stimulating the pituitary gland to release gonadotrophins is well known but has never been developed to a state of practical utility. This interference results in rendering the treated mammal infertile for various periods of time. The antibodies have been supplied by the treated mammal by active immunization or they have been supplied by passive immunization. The latter technique is of interest for short term sterilization and has been described in U.S. Pat. No. 4,676,981. However, foreign antibodies typically have a rather limited half life in the host mammal and then retreatment is necessary if the suppressed fertility condition is to be maintained.
Active immunization has generally been recognized as the more efficient route to longer term effects. However, GnRH is only a decapeptide so some measure is necessary to make it visible to the immune system of a mammal. Although some mixtures of GnRH with adjuvants or other materials have been able to provoke the production of appropriate antibodies the most widely used approach has been to couple the GnRH molecule to a large protein carrier molecule. A good review of this work appears in "Active immunization against LHRH in the female", a chapter by I. A. Jeffcoate and B. J. Keeling at pages 363 to 377 of Immunological Aspects of Reproduction in Mammals edited by D. G. Crighton and published by Butterworth's of London in 1984. This review discusses the use of various coupling agents including carbodiimide (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide), mixed anhydride, glutaraldehyde and bisdiazotized benzidene. Although the bis-diazotization coupling route involves a two step procedure such as is described in "Production and Characterization of an Antiserum to Synthetic Gonadotropin-Releasing Hormone" by Y. Koch et al in volume 55, number 3 (1973) of Biochemical and Biophysical Research Communications it is based on addition to the normally occurring side groups of the histidine or tyrosine residues.
The art has also recognized the value of using heterobifunctional coupling agents with haptens which have appropriate side groups. One approach was to use coupling agents carrying a group reactive with sulfhydryl groups and a group reactive with amino groups with haptens which carry sulfhydryl groups. In some cases haptens which do not carry such groups have been thiolated. A procedure of using such heterobifunctional agents is described in "A Method For Preparing beta-hCG COOH Peptide-Carrier Conjugates of Predictable Composition" by A. C. J. Lee et al at pages 749 to 756 of volume 17 of Molecular Immunology published by Pergamon Press of the U.K. in 1980. It involves a two step procedure of reacting a carrier protein which has many amino groups with the coupling agent (such as 6-maleimido caproic acyl N-hydroxy succinimide ester or MCS) and then reacting the so activated carrier with the sulfhydryl carrying peptide.
However, the art does not teach a technique of actively immunizing mammals against GnRH using commercially acceptable adjuvants. Most work done with the known GnRH-carrier conjugates has involved adjuvants such as Freunds' Complete or Incomplete Adjuvant which have limited utility outside the laboratory. This is believed to be due to the fact that the immunogenicity of these conjugates has been variable and to some extent not precisely controllable. This in turn is believed to arise from difficulties in developing a reproducible conjugation regimen.
The present invention involves the discovery of how to make the reproducible and controllable conjugation regimens available with heterobifunctional coupling agents available to GnRH carrier conjugates.